JP2021175393A - Photo-controlled amplification reagent, photo-controlled amplification method and detection method for target nucleic acid - Google Patents

Abstract

To provide an amplification reagent for a target nucleic acid containing a photoresponsive deoxynucleoside triphosphate that can control the initiation of the amplification reaction of the target nucleic acid by light irradiation, and a photo-controlled amplification method for the target nucleic acid using the amplification reagent.SOLUTION: The above problem is solved by an amplification reagent for a target nucleic acid containing: an enzyme having RNA-dependent DNA polymerase activity; an enzyme having DNA-dependent DNA polymerase activity; an enzyme having ribonuclease H (RNase H) activity; an enzyme having DNA-dependent RNA polymerase activity; a first primer having a sequence complementary to a part of the target nucleic acid; a second primer having a sequence homologous to a part of the target nucleic acid; ribonucleoside triphosphate, and deoxynucleoside triphosphate, with a proviso that one of the first primer and the second primer has a promoter sequence of the enzyme having RNA polymerase activity at 5'end thereof. The followings are satisfied: (1) a deoxyribonucleotide at 3'end of the primer is conjugated with a photodegradable protective group, and/or (2) at least one of deoxyadenosine triphosphate, and deoxythymidine triphosphate, deoxyguanosine triphosphate and deoxycytidine triphosphate which constitute the deoxynucleoside triphosphate is a photoreactive deoxynucleoside triphosphate conjugated with a photodegradable protective group.SELECTED DRAWING: None

Description

The present invention relates to a photo-controlled amplification reagent for a target nucleic acid containing a photoresponsive deoxynucleoside triphosphate, a photo-controlled amplification method for the target nucleic acid that initiates an amplification reaction for the target nucleic acid by irradiation with light, and a method for detecting the target nucleic acid. ..

In genetic testing used in clinical diagnosis, highly sensitive and well-reproducible measurement is realized by amplifying a very small amount of target nucleic acid contained in a clinical sample to increase the signal intensity. Examples of the amplification method when the target nucleic acid is DNA include a PCR (polymerase chain reaction) method. Further, as an amplification method when the target nucleic acid is RNA, NASBA (Nucleic Acid Sequence-Based Amplification) method (Patent Documents 1 and 2, Non-Patent Document 1), TMA (Transcription Prepared Amplification) method (Patent Document 3). , TRC (Transcription-Reverse Transcription Concentrated) method (Patent Document 4 and Non-Patent Document 2) and the like have been reported. Since the RNA amplification method can react at a relatively low temperature (for example, 41 ° C. to 46 ° C.) at a constant temperature, it can be easily applied to automation. On the other hand, the PCR method, which requires rapid temperature rise and fall, was not easy to apply to automation for the purpose of mass processing, but by adding an enzyme having chain substitution activity, the temperature was kept constant at a relatively low temperature. DNA amplification under temperature is also possible (Patent Document 5). However, in the above-mentioned reaction capable of amplifying the target nucleic acid at a relatively low temperature at a constant temperature, the reaction may proceed even at room temperature.

A digital NASBA method (Patent Document 6) can be mentioned as a technique for accurately quantifying a small amount of target RNA contained in a sample, but as described above, the amplification reaction may proceed even at room temperature, that is, After preparing the reaction solution, the amplification reaction may proceed before it is distributed to the micro-compartment, so highly accurate quantification was extremely difficult. Therefore, in order to detect a very small amount of target RNA present in a sample with higher efficiency and reproducibility, it is necessary to control the amplification reaction especially at room temperature.

As a method for controlling the amplification reaction of the target nucleic acid, a method of suppressing the amplification reaction by light irradiation using a primer or a substrate in which dNTP (deoxyribonucleoside triphosphate), which is a nucleic acid component, is protected with a photodegradable protecting group. (Non-Patent Documents 3 and 4). However, so far, photoresponsive dNTP in which the hydroxy group at the 3'position of dNTP, which is a constituent of nucleic acid, is protected by a photodegradable protecting group has been used as a constituent raw material of the target nucleic acid, and the amplification reaction is started by light irradiation. Such a photocontrolled amplification method of the target nucleic acid has not been reported.

Japanese Unexamined Patent Publication No. 2-005864 Special Table No. 4-503451 Special Table No. 4-507579 Japanese Unexamined Patent Publication No. 2000-014400 Japanese Unexamined Patent Publication No. 2015-116136 Published in the United States 2014/0141502

de Baar, M.D. P. et al. , Journal of Clinical Microbiology, 39, 1895-1902 (2001) Ishiguro, T.I. et al. , Analytical Biochemistry, 314, 77-86 (2003) Jian, W. et al. , Proc. Natl. Acad. Sci. USA, 104, 42, 16462-16467 (2007) Weidong, W. et al. et al. , Nucleic Acids Research, 35, 19, 6339-6349 (2007)

The present invention has been made based on the above circumstances, and an object of the present invention is to provide an optical control amplification method for a target nucleic acid, which initiates an amplification reaction of a trace amount of the target nucleic acid present in a sample by light irradiation.

As a result of intensive studies to solve the above problems, the present inventors have conducted a photo-controlled amplification reagent for a target nucleic acid and its amplification, which contains a photoresponsive dNTP whose initiation of an amplification reaction for the target nucleic acid can be controlled by light irradiation. We have found that the above-mentioned problems can be solved by a method for photocontrolling and amplifying a target nucleic acid using a reagent, and have completed the present invention.

That is, the present invention includes the embodiments shown in the following [1] to [8].

[1] An enzyme having RNA-dependent DNA polymerase activity, an enzyme having DNA-dependent DNA polymerase activity, an enzyme having ribonuclease H (RNase H) activity, an enzyme having DNA-dependent RNA polymerase activity, and a target nucleic acid. A first primer having a sequence complementary to a part of the target nucleic acid, a second primer having a sequence homologous to a part of the target nucleic acid, ribonucleoside triphosphate, and deoxynucleoside triphosphate. With the amplification reagent of the target nucleic acid (however, the promoter sequence of the enzyme having RNA polymerase activity is added to either one of the first primer and the second primer on the 5'terminal side thereof). There,
Photocontrolled amplification reagent of the target nucleic acid satisfying at least one of the following <1> and <2>:
<1> The primer represented by the following general formula (0), wherein the first and / or the second primer is a primer in which the deoxyribonucleotide at the 3'end of the primer is modified with a photodegradable protecting group.

In the general formula (0), Z represents an oligodeoxyribonucleotide constituting the first or second primer, and X represents a nucleobase according to any of the following formulas (2) to (5).

And Y is an o-nitrobenzyl group represented by the following general formula (7).

(In the general formula (7), X ¹ , X ² , X ³ and X ⁴ independently represent a hydrogen atom and an alkyl group or a hydroxymethyl group having 1 to 6 carbon atoms, respectively.)
Alternatively, it represents a 3- (dialkylamino) benzyl group represented by the following general formula (6).

(In general formula (6), R ¹ and R ² each independently represent an alkyl group having 1 to 8 carbon atoms, or R ¹ and R ² are integrated with the nitrogen atom to which they are bonded to 5 to 7. A heterocyclic ring of members may be formed. R ³ and R ⁴ independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a phenyl group.)
<2> At least one of deoxyadenosine triphosphate, deoxythymidine triphosphate, deoxyguanosine triphosphate and deoxycytidine triphosphate constituting the deoxynucleoside triphosphate is a photodegradable protective group. A modified photoresponsive deoxynucleoside triphosphate represented by the following general formula (1).

In the general formula (1), X represents any of the nucleobases of the following formulas (2) to (5).

And Y is an o-nitrobenzyl group represented by the following general formula (7).

(In the general formula (7), X ¹ , X ² , X ³ and X ⁴ independently represent a hydrogen atom and an alkyl group or a hydroxymethyl group having 1 to 6 carbon atoms, respectively.)
Alternatively, it represents a 3- (dialkylamino) benzyl group represented by the following general formula (6).

(In general formula (6), R ¹ and R ² each independently represent an alkyl group having 1 to 8 carbon atoms, or R ¹ and R ² are integrated with the nitrogen atom to which they are bonded to 5 to 7. A heterocyclic ring of members may be formed. R ³ and R ⁴ independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a phenyl group.)
[2] The photocontrolled amplification reagent for a target nucleic acid according to [1], wherein the target nucleic acid is DNA and / or RNA.

[3] A method for amplifying a target nucleic acid using the photo-controlled amplification reagent of the target nucleic acid according to [1] or [2], wherein the amplification reaction of the target nucleic acid is started by light irradiation. Nucleic acid amplification method.

[4] The method for amplifying a target nucleic acid according to [3], wherein the target nucleic acid is RNA.

[5] The method for amplifying RNA according to [4], wherein the method for amplifying RNA is any of the NASBA method, the TMA method, and the TRC method.

[6] The RNA amplification method according to [5], wherein the RNA amplification method is the TRC method.

[7] The photocontrolled amplification reagent according to [1] or [2] and an oligonucleotide probe whose fluorescence characteristics change when a complementary double strand is formed with a part of the amplified target nucleic acid as compared with that before the formation. A reagent for detecting the target nucleic acid.

[8] A method for amplifying a target nucleic acid using the target nucleic acid detection reagent according to [7], characterized in that the amplification reaction of the target nucleic acid is started by light irradiation and the amplified target nucleic acid is detected. A method for detecting a target nucleic acid.

By using the photo-controlled amplification reagent of the target nucleic acid of the present invention, the amplification reaction of a trace amount of the target nucleic acid present in the sample can be controlled by light irradiation. That is, in the amplification reaction of the target nucleic acid contained in the sample at a relatively low temperature at a constant temperature, the start of the nucleic acid amplification reaction can be controlled by light, and highly accurate quantification of the target nucleic acid, which has been difficult in the past, can be performed. Can be done.

Hereinafter, the present invention will be described in detail.

<1> Photo-controlled amplification reagent of the present invention Photoresponsive deoxynucleoside triphosphate represented by the general formula (1) (hereinafter, may be referred to as photoresponsive dNTP (1)) and / or the general formula ( The photocontrolled amplification reagent of the target nucleic acid of the present invention containing the primer modified with the photoresponsive deoxyribonucleotide represented by 0) is an amplification reaction of the target nucleic acid contained in the sample at a relatively low temperature at a constant temperature. By using in, the initiation of the nucleic acid amplification reaction can be controlled by light.

One aspect of the optical control amplification reagent of the present invention is a reagent containing the following components (A) to (I). The promoter sequence of (G) below is added to either one of the following (A) and (B) on the 5'end side thereof. Further, another more preferable component may be added for reasons other than the reason that it is necessary for the commonly used amplification reaction.
(A) First single-stranded oligonucleotide (first primer) having a sequence complementary to a part of the target nucleic acid,
(B) A second single-stranded oligonucleotide (second primer) having a sequence homologous to a part of the target nucleic acid,
(C) An enzyme having RNA-dependent DNA polymerase activity,
(D) Photoresponsive dNTP (1),
(E) An enzyme having ribonuclease H (RNase H) activity,
(F) An enzyme having DNA-dependent DNA polymerase activity,
(G) An enzyme having DNA-dependent RNA polymerase activity,
(H) Deoxyribonucleoside triphosphate (dNTPs: dATP, dTTP, dGTP, dCTP). However, dNTP having the same nucleobase as the photoresponsive dNTP (1) described in (D) may not be contained, or may be contained to the extent that the amplification reaction does not start before light irradiation.
(I) Ribonucleoside triphosphate (NTPs: ATP, UTP, GTP, CTP).

As described above, either one of (A) and (B) has the promoter sequence of (G) added to the 5'terminal side thereof. An enhancer sequence consisting of several to several tens of nucleotides may be inserted between the added primer and the promoter sequence.

The enzymes (C), (E) and (F) may use different enzymes, or some or all of them may be common enzymes. For example, avian myeloblastoma virus (AMV) reverse transcriptase is an enzyme that includes all of the enzymatic activities of (C), (E) and (F), and is a particularly preferable embodiment as a photoregulated amplification reagent.

Examples of the enzyme (G) include T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase. As the promoter sequence to be added to either (A) or (B), a sequence corresponding to the component (polymerase) used in (G) may be added.

The photoresponsive dNTP (1) of (D) is a deoxyribonucleoside triphosphate represented by the following general formula (1).

In the present invention, the photoresponsive dNTP (1) is assumed to contain a chemically acceptable salt, which is represented by the general formula (1).

X is a nucleobase of any of an adenine base (2), a guanine base (3), a thymine base (4), and a cytosine base (5).

Y is a photodegradable protecting group that is deprotected by irradiation with a specific wavelength, and is an o-nitrobenzyl group represented by the following general formula (7).

(In the general formula (7), X ¹ , X ² , X ³ and X ⁴ independently represent a hydrogen atom and an alkyl group or a hydroxymethyl group having 1 to 6 carbon atoms, respectively.)
Alternatively, it represents a 3- (dialkylamino) benzyl group represented by the following general formula (6).

In the general formula (6), R ¹ and R ² each independently represent an alkyl group having 1 to 8 carbon atoms. R ¹ and R ² may be integrated with the nitrogen atom to which they are bonded to form a 5- to 7-membered heterocycle. Further, R ³ and R ⁴ independently represent a hydrogen atom and an alkyl group or a phenyl group having 1 to 6 carbon atoms, respectively.

^{The alkyl group having 1 to 8 carbon atoms represented by R 1} and R ² in the general formula (6) may be linear, branched or cyclic, and may be a methyl group, an ethyl group or a propyl group. Examples thereof include a group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a hexyl group, a heptyl group and an octyl group. Further, R ¹ and R ² may form a heterocycle having a 5- to 7-membered ring together with the nitrogen atom to which they are bonded, and the carbon atom of the heterocycle formed at this time is a nitrogen atom. And may be replaced with at least one heteroatom selected from the group consisting of oxygen atoms. Examples of the heterocycle include pyrrolidine, piperidine, and morpholine.

The alkyl groups having 1 to 6 carbon atoms represented by X ¹ , X ² , X ³ and X ^{4 in the} general formula (7) and R ³ and R ^{4 in the general formula (6) are linear.} It may be branched or cyclic, and may be a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, Hexyl groups can be exemplified.

Examples of the o-nitrobenzyl group represented by the general formula (7) include an o-nitrobenzyl group, a 4- (hydroxymethyl) -2-nitrobenzyl group, a 3- (hydroxymethyl) -2-nitrobenzyl group, and 2 -(Hydroxymethyl) -6-nitrobenzyl group, 5- (hydroxymethyl) -2-nitrobenzyl group, 4-methyl-2-nitrobenzyl group, 3-methyl-2-nitrobenzyl group, 2-methyl-6 -Nitrobenzyl group, 5-methyl-2-nitrobenzyl group, 4-ethyl-2-nitrobenzyl group, 3-ethyl-2-nitrobenzyl group, 2-ethyl-6-nitrobenzyl group, 5-ethyl-2 A nitrobenzyl group can be exemplified.

Examples of the 3- (dialkylamino) benzyl group represented by the general formula (6) include 3- (dimethylamino) benzyl group, 3- (diethylamino) benzyl group, 3- (dipropylamino) benzyl group, and 3-(. Dibutylamino) benzyl group, 3- (dioctylamino) benzyl group, 3- (ethylmethylamino) benzyl group, 3- (ethylpropylamino) benzyl group, 3-pyrrolidinobenzyl group, 3-piperidinobenzyl group, 1- [3- (dimethylamino) phenyl] ethyl group, 1- [3- (diethylamino) phenyl] ethyl group, [3- (dimethylamino) phenyl] (phenyl) methyl group, [3- (diethylamino) phenyl] Examples thereof include (phenyl) methyl group, 1- [3- (dimethylamino) phenyl] -1-methylethyl group, and 1- [3- (diethylamino) phenyl] -1-methylethyl group.

Y is preferably an o-nitrobenzyl group or a 3- (diethylamino) benzyl group because of its high photodegradability.

The photoresponsive dNTP (1) of (D) provides unmodified deoxynucleoside triphosphate (dNTP) that can be used as a substrate for DNA elongation upon light irradiation. The photoresponsive dNTP (1) used is four kinds of nucleobases represented by the formulas (2) to (5) (adenine base (2), guanine base (3), thymine base (4), or cytosine base ( It may be any one of the photoresponsive dNTPs (1) having 5)), any two or three, or all.

The primer (A) and / or the primer (B) is a primer represented by the following general formula (0) in which the deoxyribonucleotide at the 3'end of the primer is modified with a photodegradable protecting group. May be good.

In the general formula (0), Z represents an oligodeoxyribonucleotide that constitutes the first or second primer, and X is an adenine base (2), a guanine base (3), a thymine base (4), and a cytosine base ( It is any nucleobase of 5), and Y represents an o-nitrobenzyl group represented by the general formula (7) or a 3- (dialkylamino) benzyl group represented by the general formula (6). ..

When the photocontrolled amplification reagent of the present invention contains the primer represented by the above general formula (0), the above (D) (photoresponsive dNTP (1)) may or may not be contained. However, when the (D) is not contained, the (H) (deoxynucleoside triphosphate) must include all four types (dATP, dTTP, dGTP, dCTP).

Next, a method for producing the photoresponsive deoxyribonucleoside triphosphate (1) used in the present invention will be described.

For example, Proceedings of the National Academia of Sciences of the United States of America, 104, 16462-16467 (2007) and Nucleic Acids Research (2007) and Nucleic Acids Reseach, 35, 6339-6349 The deoxyribonucleoside triphosphate (1) used in the present invention can be produced by triphosphorylating the hydroxy group of the deoxyribonucleoside (7) using 7) as a raw material.

(In the formula, X and Y have the same meanings as described above.)
That is, a cyclic compound (8) is obtained by reacting deoxyribonucleoside (7) with salicyl chlorophosphite in the presence of a base and then treating with pyrophosphate (step-A). Then, by reacting this with an oxidizing agent in the presence of water (step-B), deoxyribonucleoside triphosphate (1) can be produced.

The cyclic compound (8) obtained after the step-A may be isolated and purified, or may be subjected to the next step-B without being isolated and purified as it is.

In the present invention, the cyclic compound (8) is assumed to contain a chemically acceptable salt thereof, and is represented by the general formula (8).

Salicyl chlorophosphite used in this production method is commercially available.

Examples of the base that can be used in this production method include organic bases such as triethylamine, tributylamine, pyridine, and picoline.

Examples of the pyrophosphate that can be used in this production method include bis (tetrabutylammonium) dihydrogen pyrophosphate and bis (tributylammonium) pyrophosphate. These are commercially available.

Iodine can be exemplified as an oxidizing agent that can be used in this production method.

This production method may be carried out in a solvent as long as it does not inhibit the reaction. Examples of the solvent that can be used in this production method include ether solvents such as tetrahydrofuran, diethyl ether, 1,4-dioxane and 1,2-dimethoxyethane, hydrocarbon solvents such as hexane, pentane and cyclohexane, benzene and toluene. , Aromatic hydrocarbon solvents such as xylene, halogenated hydrocarbon solvents such as dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, acetonitrile, dimethylsulfoxide, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, pyridine Can be exemplified, and two or more kinds of these solvents may be mixed and used. Of these, dimethylformamide is preferably used because of its good yield.

The molar ratio of deoxyribonucleoside (7) used in this production method to salicyl chlorophosphite is preferably in the range of 1: 0.8 to 1:10, and among them, the yield is good, from 1: 1 to 1: 2. Is even more preferable.

The molar ratio of the deoxyribonucleoside (7) used in the present production method to the base is preferably in the range of 1: 0.8 to 1:10, and more preferably 1: 1 to 1: 2 in terms of good yield.

The molar ratio of deoxyribonucleoside (7) to pyrophosphate used in this production method is preferably in the range of 1: 0.8 to 1:10, and is further preferably 1: 1 to 1: 2 in terms of good yield. preferable.

The molar ratio of the deoxyribonucleoside (7) used in this production method to the oxidizing agent is preferably in the range of 1: 0.8 to 1:20, and more preferably 1: 1 to 1: 5 in terms of good yield. ..

The molar ratio of deoxyribonucleoside (7) to water used in this production method is not particularly limited, and may be used in an equal amount or more with respect to deoxyribonucleoside (7), and is preferably used as a solvent.

The reaction temperature of this production method can be appropriately selected from the range of −78 ° C. or higher and 150 ° C. or lower. Above all, it is preferable that the yield is in the range of 0 ° C. or higher and 120 ° C. or lower in terms of good yield.

The deoxyribonucleoside triphosphate (1) obtained by this production method can be purified from the reaction solution after completion of the reaction, if necessary. The purification method is not particularly limited, but the desired product can be purified by a general-purpose method such as solvent extraction, silica gel column chromatography, preparative thin layer chromatography, preparative liquid chromatography, recrystallization or sublimation. can.

Next, the preparation of the primer represented by the general formula (0), wherein the primer of the above (A) and / or the above (B) is the deoxyribonucleotide at the 3'end of the primer modified with a photodegradable protecting group. The method will be described.

For example, a deoxyribonucleotide nucleoside that can be synthesized with reference to the method disclosed in Proceedings of the National Academia of Sciences of the United States of America, 104, 16462-16467 (2007) as a raw material is used as a raw material for deoxyribonucleosides of 3'terminals. A primer represented by the general formula (0) modified with a degradable protecting group can be synthesized. The method for synthesizing a primer using deoxyribonucleoside (9) whose nucleobase is a thymine base as a raw material is described below.

Deoxyribonucleoside (9) whose 5'position is protected by a DMTr (4,4'-dimethoxytritylyl) group is bromide 4-[[(t-butyldimethylsilyl) oxy] methyl]-in the presence of a base. After reacting with 2-nitrobenzyl (10), deprotection of the benzoyl group and TBS (tert-butyldimethylsilyl) group is performed to deoxyribonucleoside (11) in which the 3'position is modified with a photodegradable protecting group. ) Is obtained. Subsequently, the hydroxymethyl group substituted with the photoreactive protecting group was reacted with succinic anhydride to introduce a carboxyl group, and the carboxyl group was changed to 3,4-dihydro-3-hydroxy-4-oxo-1, It can be converted to the activated ester (12) using 2,3-benzotriazine or the like. By reacting the obtained activated ester (12) with an amine (13) immobilized on a carrier such as polystyrene or silica gel (for example, Amino-SynBase CPG 1000/110 [LGC LINK Technologies]), 3 An immobilized deoxyribonucleoside (14) is obtained in which the'position is modified with a photodegradable protecting group. By subjecting the obtained immobilized deoxyribonucleoside (14) to a conventional automatic DNA synthesis method, a primer in which the 3'-terminal deoxyribonucleotide is modified with a photodegradable protecting group can be synthesized.

<2> Photo-Controlled Amplification Method of Target Nucleic Acid Next, the photo-controlled amplification reaction of the target nucleic acid of the present invention using the photo-controlled amplification reagent of the present invention will be described.

The target nucleic acid may be DNA or RNA having an arbitrary base sequence, and can be arbitrarily determined as long as the above (A) and (B) contain a sequence portion specific to the extent that it can be distinguished from other nucleic acids. .. The origin of the target nucleic acid is not particularly limited, and may be, for example, a polynucleotide synthesized by a nucleic acid amplification reaction typified by the PCR method, or a chemically synthesized oligonucleotide. It may be a polynucleotide extracted from blood, tissue, cells or the like, or it may be a polynucleotide extracted from food, soil, wastewater or the like.

When the target nucleic acid is DNA, a PCR method or the like can be used as a method for amplifying the target nucleic acid.

When the target nucleic acid is RNA, the target nucleic acid can be amplified by NASBA (Nucleic Acid Sequence-Based Amplification) method, TMA (Transcription Measured Amplification) method, TRC (Transcription-Revolution) method, or the like. .. In these methods, a primer containing a promoter sequence for a target RNA, reverse transcriptase and ribonuclease H (RNase H) are used to generate double-stranded DNA containing the promoter sequence, and a specific base sequence is obtained by RNA polymerase. An RNA containing the RNA is generated, and thereafter, a linkage reaction is performed using the produced RNA as a template for double-stranded DNA synthesis containing the promoter sequence.

Although the solvent used for the amplification reaction of the target nucleic acid is not particularly limited, it is preferable to use a buffer solution. Examples of the buffer solution that can be used include Tris-hydrochloride buffer solution, sodium acetate buffer solution, HEPES (2- [4- (2-HydroxyEthyl) -1-Piperazinyl] EthaneSulphonic acid) -KOH buffer solution, and sodium phosphate buffer solution. , Potassium phosphate buffer can be exemplified. Inorganic salts or carboxylic acid salts may be added to the above-mentioned buffer solution at any concentration. Examples of the inorganic salt include sodium chloride, magnesium chloride, potassium chloride, sodium bromide, magnesium bromide, potassium bromide, sodium iodide, and potassium iodide, and examples of the carboxylate include magnesium acetate. , Manganese acetate can be exemplified. Two or more of these inorganic salts and carboxylates may be mixed and used. The concentration of the inorganic salt or carboxylate added is preferably in the range of 0M or more and 5M or less. Further, an organic solvent miscible with the buffer solution may be added at a ratio of 0% or more and 80% or less. Examples of the organic solvent include dimethyl sulfoxide (DMSO), methanol, ethanol, hexamethylphosphoric acid triamide, N, N-dimethylformamide, N, N-dimethylacetamide, 1,4-dioxane, tetrahydrofuran, ethylene glycol, and glycerin. Two or more of these organic solvents may be mixed and used.

In the method for amplifying a target nucleic acid of the present invention, the amplification reaction is started by mixing the photo-controlled amplification reagent of the target nucleic acid and the target nucleic acid and performing light irradiation. That is, when the photoresponsive dNTP (1) is contained as a free deoxyribonucleoside triphosphate, in the absence of light irradiation, the dNTP having the same nucleobase as the photoresponsive dNTP (1) is contained in the amplification reaction system. Does not exist in. Therefore, even if the target nucleic acid is added to the photocontrolled amplification reagent in this state, the amplification reaction does not proceed. On the other hand, when the photoresponsive dNTP (1) is irradiated with light, the photoresponsive protecting group is released so that unmodified dNTP, which is one of the substrates for DNA elongation, is present in the photoregulated amplification reagent. Become. Therefore, by adding the target nucleic acid to the photocontrolled amplification reagent in this state and setting the reagent to appropriate amplification reaction conditions (appropriate temperature, etc.), the amplification reaction of the target nucleic acid proceeds.

Further, when the photoresponsive dNTP (1) is contained as a deoxyribonucleotide (that is, an embodiment of the general formula (0)) constituting the first and / or second primers, the photoresponsive dNTP (1) is not irradiated with light. Since the photodegradation protective group modified in (1) inhibits hybridization between the primer and the target nucleic acid, the amplification reaction does not proceed even if the target nucleic acid is added. On the other hand, when the photoresponsive dNTP (1) (general formula (0)) is irradiated with light, the photoresponsive protecting group is released and the primer can hybridize with the target nucleic acid. Therefore, by setting the photocontrolled amplification reagent to which the target nucleic acid is added under appropriate amplification reaction conditions (appropriate temperature, etc.), the amplification reaction of the target nucleic acid proceeds.

When the photoresponsive dNTP represented by the general formula (1) or the Y of the primer represented by the general formula (0) is an o-nitrobenzyl group (general formula (7)), light having a wavelength of 200 nm or more and 450 nm or less. By irradiating with, deprotection of the nitrobenzyl group proceeds. From the viewpoint of high photodecomposition rate, it is preferable to use light having a wavelength of 300 nm or more and 400 nm or less. When Y of the primer represented by the photoresponsive dNTP (1) or the general formula (0) is a 3- (dialkylamino) benzyl group, 3- (dialkyl) is obtained by irradiating with light having a wavelength of 200 nm or more and 400 nm or less. Deprotection of the amino) benzyl group proceeds. From the viewpoint of high photodecomposition rate, it is preferable to use light having a wavelength of 300 nm or more and 340 nm or less. The irradiance of light is not particularly limited, but it is preferable to use light in the range of ^{0.1 mW / cm 2} or more and 3 W / cm ^{2 or less.} The irradiation time of light is not particularly limited, but the irradiation time of 30 minutes or less is preferable, and the irradiation time of 5 minutes or less is more preferable.

The temperature at which the amplification reaction of the target nucleic acid is carried out may be appropriately set from the range of 0 ° C. or higher and 80 ° C. or lower in consideration of the composition of the solvent to be used. When the amplification reaction is carried out by the TRC method, it is preferable to carry out the amplification reaction in the range of 40 ° C. or higher and 50 ° C. or lower.

<3> Detection Method of the Present Invention In the method for amplifying the target nucleic acid of the present invention, the amplified target nucleic acid can be detected by the following method.

The target nucleic acid amplified by the photocontrolled amplification reagent of the present invention may be detected by adding a detection component in advance or after the amplification reaction and measuring the fluorescence intensity or chemiluminescence intensity derived from the component. A preferred embodiment of the detection component is an oligonucleotide probe whose fluorescence characteristics change when a double strand complementary to a part of the target nucleic acid is formed as compared with that before the formation.

An example of the probe is DNA bound with an intercalator fluorescent dye having a sequence complementary or homologous to a part of the target nucleic acid. The sequence of the DNA portion needs to be a sequence existing in the target nucleic acid and complementary or homologous to a portion sufficiently distinguishable from a nucleic acid other than the target nucleic acid. The length of the DNA portion is preferably 6 nucleotides or more and 100 nucleotides or less, more preferably 10 nucleotides or more and 30 nucleotides or less for specific analysis of the target nucleic acid. The DNA portion has a non-target nucleic acid at the 3'end so that extension from the 3'end by an enzyme having RNA-dependent DNA polymerase activity does not occur when a complementary bond is formed with the amplified target nucleic acid. It is preferred that a complementary sequence is added or that the 3'end is chemically modified (eg, amination).

In the intercalating fluorescent dye, when the above-mentioned DNA portion forms a complementary bond with another nucleic acid, it intercalates into the double-stranded portion and the fluorescence characteristics change. For this purpose, for example, the intercalating fluorescent dye may be attached to the DNA via a suitable molecular length linker that does not interfere with the intercalation to the double-stranded moiety. The linker is not particularly limited as long as it is a molecule that does not prevent the intercalating fluorescent dye from intercalating into the double-stranded portion. In particular, a linker molecule selected from bifunctional hydrocarbons having functional groups at both ends is convenient and preferable for modifying an oligonucleotide. Alternatively, a commercially available reagent set (for example, C6-Thiolmodifer manufactured by Clontech) may be used.

The intercalating fluorescent dye is not particularly limited as long as its fluorescent characteristics change, for example, the emitted fluorescence wavelength fluctuates by intercalating into a double strand, but it is easy to measure. From the above viewpoints, those having the property of increasing the fluorescence intensity by intercalation are particularly preferable. Specifically, thiazole orange and oxazole yellow, which have a particularly remarkable change in fluorescence intensity, and derivatives thereof can be exemplified as preferable intercalator fluorescent dyes.

The position at which the intercalating fluorescent dye is bound to the DNA portion via the linker hinders the intercalation of the intercalating fluorescent dye to the double strand such as the 5'end, 3'end or the central portion of the DNA portion. There is no particular limitation as long as it is not possible and the complementary binding between the DNA portion and RNA is not inhibited.

Another example of an oligonucleotide probe whose fluorescence characteristics change when it forms a double strand complementary to a part of the target nucleic acid as compared with that before the formation is a molecular beacon. A molecular beacon is a DNA having a sequence complementary or homologous to a part of a target nucleic acid, and has a stem-loop structure having a fluorescent dye and a quencher at both ends thereof. In the state of the stem-loop structure, the fluorescence of the fluorescent dye is suppressed by the quencher, but when the region complementary to the target RNA present in the loop sequence hybridizes with the amplification product generated during the reaction, the stem portion is cleaved. It emits fluorescence. Stems can be formed by forming double strands of DNA in the molecule and introducing a pair of highly associative fluorescent dye (Cy3, etc.) and quencher (azo compound, etc.) into the DNA.

Another example of an oligonucleotide probe whose fluorescence characteristics change when it forms a double strand complementary to a part of the target nucleic acid as compared with that before the formation is a FRET (Fluorescence Resonance Energy Transfer) probe. In this case, in the sequence of the target nucleic acid, two probes designed inside the first and second primers used for amplification are used. Of the two probes, the 3'end of one probe is modified with a donor fluorescent dye, and the 5'end of the other probe is modified with an acceptor fluorescent dye. By designing the donor fluorescent dye and the acceptor fluorescent dye to be close to each other when hybridized, the target nucleic acid is detected by the FRET phenomenon caused by the hybridization of the two probes with the amplification product generated during the reaction. can.

Yet another example of an oligonucleotide probe whose fluorescence properties change when it forms a double strand complementary to a portion of the target nucleic acid as compared to before formation is the TaqMan probe. In this case, in the sequence of the target nucleic acid, one probe designed inside the first and second primers used for amplification is used. A fluorescent dye (reporter dye) is labeled at the 5'end of the TaqMan probe, and a quencher is labeled at the 3'end. The fluorescence of the reporter dye is suppressed by the quencher, but if the probe hybridizes to the target RNA during the primer extension reaction by DNA polymerase, the probe is degraded by 5'→ 3'exonuclease activity, and the reporter It can be detected by the fluorescence generated by the liberation of the dye.

Next, the present invention will be described in more detail with reference to Examples and Synthesis Examples, but the present invention is not limited thereto. The reagents and anhydrous solvents used in the reaction were commercially available products (manufactured by Tokyo Chemical Industry Co., Ltd., manufactured by Kanto Chemical Co., Ltd., manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., manufactured by Sigma-Aldrich) without purification. Nuclear magnetic resonance (NMR) spectra were measured on 400 MHz Bruker AVANCE III 400 and 400 MHz Bruker AVANCE III HD. The chemical shift value was shown in ppm using tetramethylsilane (TMS) as an internal standard substance. s stands for singlet, d stands for doublet, t stands for triplet, q stands for quartet, quint stands for quintet, br s stands for broad singlet, and m stands for quartet.

Synthesis example 1

3'-O- (2-nitrobenzyl) -2'-deoxyadenosine (115 mg, 0.30 mmol) is azeotroped 5 times with dehydrated toluene (1 mL) in a 25 mL volumetric flask and then with dehydrated pyridine (1 mL). Azeotropically boiled 5 times. Under an argon atmosphere, dehydrated pyridine (2 mL) was added, and salicyl chlorophosphate (73 mg, 0.36 mmol) dissolved in 1,4-dioxane (1 mL) was added. After reacting for 30 minutes, tributylammonium pyrophosphate (198 mg, 0.36 mmol) dissolved in tributylamine (220 μL, 0.90 mmol) and DMF (N, N-dimethylformamide) (2 mL) was added, and the reaction was carried out for 1 hour. I let you. Subsequently, a 1% (w / v) iodine solution (pyridine / water = 9: 1) was added until the solution turned brown, and then the reaction was carried out for 30 minutes. Water (2 mL) was added and the mixture was further stirred for 30 minutes. After completion of the reaction, ethyl acetate (10 mL) was added and the mixture was vigorously stirred to remove the organic phase three times. The recovered aqueous phase was concentrated under reduced pressure, and the residue was purified by reverse phase column chromatography (triethylamine acetate buffer: methanol, 10: 0 → 8: 2) and then concentrated under reduced pressure. The obtained residue was dissolved in methanol (1 mL), and 0.6 M sodium perchlorate / acetone solution (10 mL) was added thereto. The solution was centrifuged to remove the supernatant. Further, acetone (10 mL) was added, and the mixture was centrifuged to remove the supernatant three times. The obtained white solid was dried under reduced pressure to obtain 3'-O- (2-nitrobenzyl) -2'-deoxyadenosine triphosphate (NB-dATP) (34 mg, 51 μmol, 17%, ^{1 1} H NMR purity 95). %) Was obtained.

_{^{1 H NMR (D 2 O,}} 400MHz): δ8.45 (s, 1H), 8.16 (s, 1H), 8.01 (m, 1H), 7.68-7.73 (m, 2H) , 7.52 (m, 1H), 6.42 (dd, J = 6.2,8.6Hz, 1H), 4.96 (m, 2H), 4.61 (m, 1H), 4.45 (m, 1H), 4.07-4.19 ( m, 2H), 2.66-2.78 (m, 2H) ppm; 31 P NMR (D 2 O, 161MHz): δ-6.29 ( m, 1P), -11.0 (m, 1P), -21.8 (m, 1P) ppm; HR-MS (ESI-TOF): magnetic resonance C ₁₇ H ₂₀ N ₆ O ₁₄ P ₃ ; [M −H ⁺ ] −: 625.0490 (calculated: 625.0250).
Synthesis example 2
N ⁶ , N ⁶ -bis (tert-butoxycarbonyl) -5'-O-tert-butyldimethylsilyl-2'-deoxyadenosin (2.88 g, 5.1 mmol) was dissolved in methylene chloride (20 mL) and iodine was added. Sodium chloride (100 mg, 0.67 mmol), 1.5 M aqueous tetrabutylammonium hydroxide solution (6.78 mL, 10 mmol), 1.0 M aqueous sodium hydroxide solution (7.63 mL, 7.6 mmol) and distilled water (10 mL). In addition, the mixture was stirred at room temperature for 5 minutes. A solution of m- (diethylamino) benzyl chloride (3.02 g, 15 mmol) in methylene chloride (9.0 mL) was added to the obtained mixed solution, and the mixture was reacted at room temperature for 15 hours under shading. The aqueous phase and the organic phase were separated, and the aqueous phase was extracted twice with methylene chloride (50 mL). The organic phases were combined and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was purified by flash silica gel column chromatography (hexane: ethyl acetate = 70:30) and N ⁶ , N ⁶ -bis (tert-butoxycarbonyl) -5'-O-tert-butyldimethylsilyl-3. '-O- [3- (diethylamino) benzyl] -2'-deoxyadenosine (625 mg, 17%) was obtained as a yellow solid.

^{1 1} H NMR (400 MHz, CDCl ₃ ): δ8.84 (s, 1H), 8.42 (s, 1H), 7.19 (t, J = 7.9 Hz, 1H), 6.64-6.61 (M, 3H), 6.55 (m, 1H), 4.53 (d, J = 1.4Hz, 2H), 4.37 (m, 1H), 4.27 (m, 1H), 3. 89 (dd, J = 11.1, 4.0Hz, 1H), 3.78 (dd, J = 11.1, 3.2Hz, 1H), 3.36 (q, J = 7.1Hz, 4H) , 2.70-2.59 (m, 2H), 1.45 (s, 18H), 1.16 (t, J = 7.1Hz, 6H), 0.88 (s, 9H), 0.07 (S, 3H), 0.06 (s, 3H).
Synthesis example 3
^{N 6} , N ⁶ -bis (tert-butoxycarbonyl) -5'-O-tert-butyldimethylsilyl-3'-O- [3- (diethylamino) benzyl] -2'-deoxy obtained in Synthesis Example 2 Weigh adenosine (625 mg, 0.87 mmol) in a light-shielding container, dissolve it in methylene chloride (30 mL), add activated silica gel (vacuum dried at 80 ° C. for 3 hours), add silica gel (6.0 g), and add the solvent under reduced pressure. Distilled away. The obtained residue was heated at 80 ° C. under reduced pressure for 13 hours. The obtained solid was washed with methanol and chloroform, and the filtrate was concentrated to concentrate 5'-O-tert-butyldimethylsilyl-3'-O- [3- (diethylamino) benzyl] -2'-deoxyadenosine. (369 mg, 81%) was obtained as a yellow solid.

^{1 1} H NMR (400 MHz, CDCl ₃ ): δ8.32 (s, 1H), 8.14 (s, 1H), 7.17 (t, J = 7.8 Hz, 1H), 6.63-6.59 (M, 3H), 6.47 (t, J = 6.5Hz, 1H), 6.05 (brs, 2H), 4.51 (d, J = 3.7Hz, 2H), 4.34 (m) , 1H), 4.24 (m, 1H), 3.88 (dd, J = 11.0.4.2Hz, 1H), 3.76 (dd, J = 11.0, 3.2Hz, 1H) , 3.33 (q, J = 7.0Hz, 4H), 2.64-2.61 (m, 2H), 1.13 (t, J = 7.0Hz, 6H), 0.89 (s, 9H), 0.06 (s, 3H), 0.05 (s, 3H).
Then, 5'-O-tert-butyldimethylsilyl-3'-O- [3- (diethylamino) benzyl] -2'-deoxyadenosine (369 mg, 0.70 mmol) was dissolved in tetrahydrofuran (3.0 mL). It was cooled to 0 ° C. A 1M tetrabutylammonium fluoride-tetrahydrofuran solution (0.70 mL, 0.70 mmol) was slowly added dropwise to the obtained mixed solution, and then the temperature was raised to room temperature. The reaction was carried out at room temperature for 2 hours, and methanol (3.0 mL) was added to stop the reaction. The reaction mixture was concentrated, and the obtained residue was purified by flash silica gel column chromatography (ethyl acetate: methanol = 90:10) to purify 3'-O- [3- (diethylamino) benzyl] -2'-deoxyadenosine ( 202 mg, 70%) was obtained as a yellow solid.

^{1 1} H NMR (400 MHz, CDCl ₃ ): δ8.32 (s, 1H), 7.81 (s, 1H), 7.20 (t, J = 8.1 Hz, 1H), 6.65-6.62 (M, 3H), 6.57 (m, 1H), 6.28 (m, 1H), 5.74 (brs, 2H), 4.54 (s, 2H), 4.49 (m, 1H) , 4.39 (s, 1H), 3.99 (m, 1H), 3.70 (m, 1H), 3.36 (q, J = 7.0Hz, 4H), 3.01 (m, 1H) ), 2.44 (m, 1H), 1.17 (t, J = 7.0Hz, 6H).
Synthesis example 4
Dehydrated pyridine (3'-O- [3- (diethylamino) benzyl] -2'-deoxyadenosine (70.0 mg, 0.17 mmol) obtained in Synthesis Example 3 twice with dehydrated toluene (3.0 mL). Water was removed by azeotropically boiling with 3.0 mL) three times. Salicyl chlorophosphate (40.0 mg, 0.20 mmol) and 1,4-dioxane (1.0 mL) were added to a dry solid dehydrated pyridine (2.0 mL) solution and reacted at room temperature for one and a half hours. rice field. Subsequently, a DMF solution (1.0 mL) of bis (tributylammonium) pyrophosphate (82.0 mg, 0.15 mmol) and tributylamine (0.12 mL, 0.50 mmol) were added, and the mixture was reacted at room temperature for 2 hours. A solution of iodine (32 mg, 0.13 mmol) / pyridine (9.0 mL) / distilled water (1.0 mL) was added until the reaction turned red. After confirming that the reaction solution did not fade even after stirring at room temperature for about 15 minutes, distilled water (2.0 mL) was added and the mixture was reacted at room temperature for 12 hours. The reaction mixture was washed with ethyl acetate (60 mL) and the aqueous phase was concentrated. The obtained residue was purified by octadecyl group-modified silica gel column chromatography (water: methanol = 9: 1), and the solvent was distilled off under reduced pressure. The obtained residue was suspended in distilled water (1.3 mL) and methanol (0.5 mL), and a solution of sodium perchlorate (850 mg) in acetone (10 mL) was added for centrifugation to remove the supernatant. The solvent was distilled off under reduced pressure. The same operation was performed twice to obtain 3'-O- [3- (diethylamino) benzyl] -2'-deoxyadenosine triphosphate (DEAB-dATP) (13 mg, 12%) as a white solid.

_{^{1 H NMR (D 2 O,}} 400MHz): δ8.14 (m, 1H), 7.56 (m, 3H), 7.53 (m, 1H), 7.41 (m, 1H), 6.40 (M, 1H), 4.60 (m, 2H, obcured by the reactive proton of D ₂ O), 4.58 (m, 1H, obcured by the reactive proton of D ₂ O), 4.40 (m, 1H) 1H), 4.11 (m, 2H ), 3.57 (m, 4H), 2.66 (m, 2H), 1.01 (m, 6H); 31 P NMR (D 2 O, 162MHz): δ-8.0 (m, 1P) , - 11.4 (d, 1P), - 21.5 (m, 1P); HR-MS (ESI-TOF): molecular formula C 21 H 30 N 6 O 12 P ₃ ; [MH ⁺ ]-: 651.1179 (calculated: 651.118).

Synthesis example 5
2-Nitro-p-xylene glycol (5.00 g, 27.3 mmol) and imidazole (3.72 g, 54.6 mmol) were dissolved in DMF (15.0 mL) under an argon atmosphere and cooled to 0 ° C. A solution of t-butyldimethylsilyl chloride (4.25 g, 27.3 mmol) in DMF (10.0 mL) was added to the obtained solution. The obtained mixed solution was heated to room temperature and reacted for 1.5 hours, then methanol (20 mL) was added, and the mixture was stirred at room temperature for 30 minutes to stop the reaction. Distilled water (200 mL) was added to the residue obtained by distilling off the solvent in the reaction solution under reduced pressure, and the mixture was extracted 5 times with ethyl acetate (200 mL). The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was purified by flash silica gel column chromatography (hexane: ethyl acetate = 4: 1) and 4-[[(t-butyldimethylsilyl) oxy] methyl] -2-nitrobenzenemethanol (1.9 g, 23). %) Was obtained as a white-yellow solid.

^{1 1} H NMR (400 MHz, CDCl ₃ ): δ8.06 (s, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 4 .93 (d, J = 6.7Hz, 2H, CH _2- OH), 4.79 (s, 2H, CH _2- OTBS), 2.51 (t, J = 6.7Hz, 1H, OH), 0.95 (s, 9H, TBS), 0.11 (s, 6H, TBS).
Synthesis example 6
4-[[(T-butyldimethylsilyl) oxy] methyl] -2-nitrobenzenemethanol (648 mg, 2.17 mmol), triphenylphosphine (1.71 g, 6.51 mmol), tetrachloride obtained in Synthesis Example 5. Carbon (2.16 g, 6.51 mmol) and diisopropylethylamine (929 mg, 7.16 mmol) were dissolved in THF (tetrahydrofuran) (7.3 mL) under an argon atmosphere, and the mixture was stirred at room temperature for 1 hour. Distilled water (50 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (100 mL). The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was purified by flash silica gel column chromatography (hexane: ethyl acetate = 15: 1) and 4-[[(t-butyldimethylsilyl) oxy] methyl] -2-nitrobenzyl bromide (747 mg, 83). %) Was obtained as a white solid.

_{^{1 H NMR (400MHz, CDCl 3}} ): δ8.00 (s, 1H), 7.55-7.51 (m, 2H), 4.82 (s, CH 2 -Br), 4.79 (s, 2H, CH _2- OTBS), 0.95 (s, 9H, TBS), 0.11 (s, 6H, TBS).
Synthesis example 7
5'-O- (4,4'-dimethoxytrityryl) thymidine (2.72 g, 5.0 mmol) was dissolved in a mixed solvent (25 mL) of dichloromethane / dimethylacetamide (10: 1) and triethylamine (607 mg, 6). .0 mmol) was added, and the mixture was stirred at room temperature for 10 minutes, benzoyl chloride (773 mg, 5.5 mmol) was added, and the mixture was reacted at room temperature for 15 hours. After the reaction, the mixture was diluted with ethyl acetate (50 mL) and washed with saturated brine (30 mL). The residue obtained by distilling off the solvent under reduced pressure was purified by flash silica gel column chromatography (toluene: ethyl acetate = 3: 1 → 1: 1) to obtain N-benzoyl-5'-O- (4, A mixture of 4'-dimethoxytrityryl) thymidine and 3'-O-benzoyl-5'-O- (4,4'-dimethoxytrityryl) thymidine was obtained (1.78 g, 55%, N-Bz form: O-Bz compound = 6.5: 1) was obtained as a colorless solid.

N-benzoyl-5'-O- (4,4'-dimethoxytritylyl) thymidine; ^{1 1} H NMR (400 MHz, CDCl ₃ ): δ7.88 (dd, J = 7.2, 1.2 Hz, 2H), 7.73 (d, J = 0.84Hz, 1H), 7.55 (t, J = 7.4Hz, 1H), 7.37-7.42 (m, 4H), 7.25-7.30 (M, 5H), 7.19-7.23 (m, 2H), 6.82 (d, J = 8.0Hz, 4H), 6.34 (t, J = 7.2Hz, 1H), 4 .49 (quint, J = 2.5Hz, 1H), 3.97 (q, J = 2.5Hz, 1H), 3.74 (s, 6H), 3.42 (dd, J = 10.6, 2.7Hz, 1H), 3.31 (dd, J = 10.6, 2.7Hz, 1H), 2.23-2.34 (m, 2H), 1.41 (s, 3H).
3'-O-benzoyl-5'-O- (4,4'-dimethoxytritylyl) thymidine; ^{1 1} H NMR (400 MHz, CDCl ₃ ): δ8.02 (t, J = 6.9 Hz, 2H), 7 .66 (d, J = 0.96Hz, 1H), 7.55 (t, J = 7.4Hz, 1H), 7.37-7.42 (m, 4H), 7.25-7.30 ( m, 5H), 7.09-7.14 (m, 2H), 6.82 (d, J = 8.0Hz, 4H), 6.50 (dd, J = 5.6Hz, 3.0Hz, 1H) ), 5.68 (d, J = 5.6Hz, 1H), 4.26 (d, J = 1.8Hz, 1H), 3.73 (s, 6H), 3.53 (t, J = 2) .4Hz, 1H), 2.47-2.64 (m, 1H), 1.37 (s, 3H).
Synthesis example 8
4-[[(T-Butyldimethylsilyl) oxy] methyl] -2-nitrobenzyl (377 mg, 1.05 mmol) obtained in Synthesis Example 6 was dissolved in methylene chloride (2.0 mL) and 10 at room temperature. After stirring for a minute, N-benzoyl-5'-O- (4,4'-dimethoxytritylyl) thymidin and 3'-benzoyl-5'-O- (4,4'-) obtained in Synthesis Example 7 were obtained. Mixture of dimethoxytritylyl) thymidin (521 mg, 0.81 mmol, N-Bz form: O-Bz form = 4: 1), tetrabutylammonium iodide (236 mg, 0.64 mmol), 1 M aqueous sodium hydroxide solution (1. A mixed solution of 62 mL, 1.62 mmol) and methylene chloride (6 mL) was added dropwise and reacted at room temperature for 3 hours. The reaction was diluted with dichloromethane (20 mL), washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the obtained residue was purified by flash silica gel column chromatography (toluene: ethyl acetate = 30: 1) to obtain a product (146 mg). Subsequently, the obtained product was dissolved in a 2M ethanol / methylene chloride solution of ammonia (16 mL, ethanol: methylene chloride = 2.5: 1), stirred at room temperature for 6.5 hours, and then the solvent was retained under reduced pressure. The residue obtained after removal was purified by flash silica gel column chromatography (hexane: ethyl acetate = 1: 1) to 3'-O- [4-[[(t-butyldimethylsilyl) oxy] methyl]-. 2-Nitro] benzyl-5'-O- (4,4'-dimethoxytritylyl) thymidin (46.7 mg, 2 steps 7%) was obtained as a colorless solid.

^{1 1} H NMR (400 MHz, CDCl ₃ ): δ8.05 (brs, 1H), 7.98 (brs, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.59 (d, J) = 4.6Hz, 2H), 7.39 (d, J = 8.6Hz, 2H), 7.21-7.31 (m, 7H), 6.83 (dd, J = 8.9, 1. 2Hz, 4H), 6.39 (dd, J = 8.4,5.6Hz, 1H), 4.85 (q, J = 14.6Hz, 2H), 4.76 (brs, 2H), 4. 33 (d, J = 5.6Hz, 1H), 4.22 (d, J = 2.4Hz, 1H), 3.78 (s, 6H), 3.52 (dd, J = 10.6, 3) .2Hz, 1H), 3.37 (dd, J = 10.6, 3.2Hz, 1H), 2.53-2.58 (m, 1H), 2.21-2.29 (m, 1H) , 1.47 (d, J = 0.8Hz, 3H), 0.95 (s, 9H), 0.12 (s, 6H).
Synthesis example 9
3'-O- [4-[[(t-butyldimethylsilyl) oxy] methyl] -2-nitro] benzyl-5'-O- (4,4'-dimethoxytritylyl) obtained in Synthesis Example 8 To a solution of timidine (157 mg, 0.19 mmol) in THF (3.8 mL) was added a 1 M solution of tetrabutylammonium fluoride in THF (0.23 mL, 0.23 mmol), and the mixture was reacted at 0 ° C. for 2 hours. It was diluted with ethyl acetate (20 mL) and washed with saturated brine (20 mL). The residue obtained by distilling off the solvent under reduced pressure was purified by flash silica gel column chromatography (hexane: ethyl acetate = 1: 1 → 1: 4) to obtain 3'-O- [4- (hydroxymethyl). -2-Nitro] Benzyl-5'-O- (4,4'-dimethoxytritylyl) thymidine (124 mg, 92%) was obtained in the form of a colorless oil.

^{1 1} H NMR (400 MHz, CDCl ₃ ): δ8.10 (d, J = 1.4 Hz, 1H), 7.91 (brs, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7 .61 (dd, J = 8.6, 1.1Hz, 2H), 7.38 (dd, J = 7.0, 1.5Hz, 2H), 7.31-7.20 (m, 7H), 6.82 (dd, J = 8.9, 3.4Hz, 4H), 6.39 (dd, J = 8.4,5.6Hz, 1H), 4.86 (q, J = 14.9Hz, 2H), 4.79 (s, 2H), 4.31 (d, J = 5.9Hz, 1H), 4.23 (d, J = 2.5Hz, 1H), 3.79 (s, 6H) , 3.53 (dd, J = 10.6, 2.7Hz, 1H), 3.35 (dd, J = 10.6, 2.7Hz, 1H), 2.54 (dq, J = 13.6) , 5.4Hz, 1H), 2.17-2.28 (m, 1H), 1.94 (t, J = 6.0Hz, 1H), 1.49 (d, J = 0.96Hz, 3H) ..
Synthesis example 10
Of 3'-O- [4- (hydroxymethyl) -2-nitro] benzyl-5'-O- (4,4'-dimethoxytritylyl) thymidine (130 mg, 0.18 mmol) obtained in Synthesis Example 9. To a solution of methylene chloride (3.6 mL), succinic anhydride (22 mg, 0.21 mmol), triethylamine (36 mg, 0.36 mmol) and 4-dimethylaminopyridine (22 mg, 0.18 mmol) were added, and the mixture was reacted at room temperature for 18 hours. rice field. It was diluted with ethyl acetate (20 mL) and washed with purified water (20 mL). The solvent was evaporated under reduced pressure to give the product (78.2 mg) in the form of a colorless oil. 3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (90 mg, 0.55 mmol) in a solution of the resulting product (21.6 mg) in dimethylformamide (3.6 mL). And 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (70 mg, 0.37 mmol) were added and reacted at room temperature for 18 hours. It was diluted with ethyl acetate (20 mL) and washed with purified water (20 mL). The residue obtained by distilling off the solvent under reduced pressure was purified by flash silica gel column chromatography (chloroform: ethyl acetate = 3: 1) to 3'-O-[[[[[4- (3,4). −Dihydro-4-oxo-1,2,3-benzotriazine) -3-yloxy] carbonyl ethyl] carbonyl] oxymethyl] -2-nitro] benzyl-5'-O- (4,4'-dimethoxytrityryl) ) Thymidine (65 mg, 38% in 2 steps) was obtained in the form of a colorless oil.

^{1 1} H NMR (400 MHz, CDCl ₃ ): δ8.36 (dd, J = 8.0, 1.0 Hz, 1H), 8.22 (dd, J = 8.2,0.48 Hz, 1H), 8. 08 (dd, J = 13.0, 1.6Hz, 1H), 8.00 (dt, J = 7.4, 1.4Hz, 2H), 7.84 (dt, J = 7.4,1. 1Hz, 1H), 7.74 (d, J = 8.0, 2.5Hz, 1H), 7.63 (dd, J = 8.0, 1.7Hz, 1H), 7.59 (d, J) = 1.1Hz, 1H), 7.38 (dd, J = 8.5, 1.5Hz, 2H), 7.30
-7.21 (m, 7H), 6.82 (dd, J = 8.9, 2.0Hz, 4H), 6.39 (dd, J = 8.4,5.6Hz, 1H), 5. 29 (s, 2H), 5.24 (s, 1H), 4.86 (q, J = 15.0Hz, 2H), 4.32 (d, J = 5.9Hz, 1H), 4.22 ( d, J = 2.4Hz, 1H), 3.78 (s, 6H), 3.52 (dd, J = 10.6, 2.7Hz, 1H), 3.36 (dd, J = 10.6) , 2.7Hz, 1H), 3.13 (t, J = 6.6Hz, 2H), 2.91 (t, J = 6.6Hz, 2H), 2.50-2.58 (m, 1H) , 2.16-2.28 (m, 1H), 1.48 (d, J = 1.0Hz, 3H).
Synthesis example 11

Amino-SynBase CPG 1000/110 (LCAA) (manufactured by LGC LINK Technologies) (400 mg, loading amount 105 μmol / g) was placed in a plastic centrifuge tube and set in a solid phase synthesizer, and obtained in Synthesis Example 103. '-O-[[[[[4- (3,4-dihydro-4-oxo-1,2,3-benzotriazine) -3-yloxy] carbonylethyl] carbonyl] oxymethyl] -2-nitro] benzyl A DMF solution of -5'-O- (4,4'-dimethoxytritylyl) thymidine (61.6 mg, 64.5 μmol) and diisopropylethylamine (8.4 mg, 64.5 μmol) was added and reacted at room temperature for 3 days. .. After completion of the reaction, the solid phase was taken out by suction filtration, washed with ethyl acetate and dried to obtain a solid phase-supported thymidine derivative (349.5 mg). The loading amount of the thymidine derivative in the solid phase was 100.5 μmol / g.

Hereinafter, the present invention will be described in more detail with reference to examples when the TRC (Transcription-Reverse Transcription Concerted) method is used as the nucleic acid amplification method, but the present invention is not limited to these examples.

Example 1 Effect of dATP concentration on TRC reaction The effect of the dATP addition concentration on the detection time in the TRC method was investigated.

(1) The standard RNA (SEQ ID NO: 1) was prepared by in vitro transcription from a plasmid into which a hepatitis C virus standard RNA (hereinafter, also simply referred to as standard RNA) gene was inserted. Diluted the standard RNA so that 10 ³ copies / 2 [mu] L with water for injection, which was used as a RNA sample.

(2) After dispensing 12 μL of the reaction solution having the following composition into a 0.5 mL volume PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), 2 μL of the RNA sample was added. The molecular beacon probe (SEQ ID NO: 2), which is a probe for detecting standard RNA, contains a part of the homologous strand of standard RNA [from 108th to 123rd of SEQ ID NO: 1]. The first primer (SEQ ID NO: 3) is a T7 promoter sequence (specifically, from 125th to 145th of SEQ ID NO: 1: SEQ ID NO: 5) on the 5'terminal side of a part of the complementary strand of standard RNA. It is an oligonucleotide to which SEQ ID NO: 6) is added.

Composition of reaction solution: The concentration is the final concentration after addition of the initiator described below (in 20 μL) 66 mM Tris-HCl buffer (pH 8.36).
0.33 mM dCTP, dGTP, dTTP, respectively
2.0 mM ATP, CTP, GTP, UTP respectively
3.3 mM ITP
150 mM trehalose 50 nM molecular beacon probe (including a part of the homologous strand of standard RNA [from 108th to 123rd of SEQ ID NO: 1]: SEQ ID NO: 2)
1.0 μM first primer (SEQ ID NO: 3)
1.0 μM second primer (part of the homologous strand of standard RNA [1st to 16th of SEQ ID NO: 1]: SEQ ID NO: 4)
12.8U AMV reverse transcriptase 166U T7 RNA polymerase dATP at various concentrations (0 to 3300 μM)
(3) The above reaction solution was kept warm at 46 ° C. for 3 minutes, and then 6 μL of an initiator having the following composition was added.

Initiator composition: The concentration is the final concentration after addition of the initiator (in 20 μL) 18.4 mM magnesium chloride 90.0 mM potassium chloride 0.1% (w / v) Tween 20 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
9.0% (v / v) DMSO
2.5% (w / v) glycerol (4) Using a fluorescence spectrophotometer with a temperature control function that can directly measure the PCR tube, react at 46 ° C. and at the same time, the fluorescence intensity of the reaction solution (excitation wavelength 470 nm, fluorescence). Wavelength 520 nm) was measured over time for 30 minutes. The time when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a predetermined time by the fluorescence intensity ratio of the background) exceeded 2.3 was defined as 0 minutes when the initiator was added, and was defined as the detection time.

The results are shown in Table 1. It can be seen that when the dATP concentration in the reaction solution is 165 μM or more, there is no effect on the amplification reaction.

Example 2 Effect of photodegradable protecting group on TRC reaction (Part 1)
The effect of photodegradable protecting groups that modify dNTPs on the TRC reaction was investigated.

(1) The 3'terminal hydroxy group of adenosine was modified with a 2-nitrobenzyl group as a photodegradable protecting group by the synthetic method described in the literature (Nucleic Acids Research, 35, 6339-6349 (2007)), 3'. -O- (2-nitrobenzyl) -2'-deoxyadenosine was synthesized.

(2) The solution containing 3'-O- (2-nitrobenzyl) -2'-deoxyadenosine synthesized in (1) was dissolved in DMSO to a constant concentration.

(3) 8 μL of the reaction solution having the following composition was dispensed into a 0.5 mL volume PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and then 2 μL of the RNA sample prepared in Example 1 (1) was added. ..

Composition of reaction solution: The concentration is the final concentration after addition of the initiator described below (in 20 μL) 66 mM Tris-HCl buffer (pH 8.36).
0.33 mM dATP, dCTP, dGTP, dTTP, respectively
2.0 mM ATP, CTP, GTP, UTP respectively
3.3 mM ITP
150 mM trehalose 50 nM molecular beacon probe (SEQ ID NO: 2)
1.0 μM first primer (SEQ ID NO: 3)
1.0 μM second primer (SEQ ID NO: 4)
12.8U AMV reverse transcriptase 166UT7 RNA polymerase (4) The above reaction solution was kept warm at 46 ° C. for 3 minutes, and then 10 μL of an initiator having the following composition was added. Although the DMSO concentration is different from that of Example 1 (9% (v / v)), it has been confirmed in advance that there is no effect on the TRC reaction.

Initiator composition: Concentration is the final concentration after addition of the initiator (in 20 μL) 18.4 mM magnesium chloride 90.0 mM potassium chloride 0.1% (w / v) Tween 20 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
2.5% (w / v) Glycerol 10% (v / v) DMSO (containing 0 to 100 μM as a photodegradable protecting group, (2)
Prepared with)
(5) The detection time was determined by measurement in the same manner as in Example 1 (4).

The results are shown in Table 2. When a 2-nitrobenzyl group (o-nitrobenzyl group) is used as the photodegradable protecting group, it can be seen that if the concentration is 50 μM or less, there is no effect on the TRC reaction.

Example 3 Effect of 2'-deoxyadenosine triphosphate (dATP) modified with a photodegradable protecting group on the TRC reaction (1) 1.65 mM 3'-O- (2-nitrobenzyl) -2'- The deoxyadenosine triphosphate (NB-dATP, synthesized in Synthesis Example 1) solution ^{was irradiated with ultraviolet light (wavelength 365 nm, intensity 700 μW / cm 2} ) for 1 to 40 minutes. As a control, an unmodified dATP solution irradiated with the light source for 40 minutes, an NB-dATP solution not irradiated with the light source, and an unmodified dATP solution were also prepared.

(2) 12 μL of the reaction solution having the following composition was dispensed into a 0.5 mL volume PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and then 2 μL of the RNA sample prepared in Example 1 (1) was added. ..

Composition of reaction solution: The concentration is the final concentration after addition of the initiator described below (in 20 μL) 66 mM Tris-HCl buffer (pH 8.36).
0.33 mM dCTP, dGTP, dTTP, respectively
2.0 mM ATP, CTP, GTP, UTP respectively
3.3 mM ITP
150 mM trehalose 50 nM molecular beacon probe (SEQ ID NO: 2)
1.0 μM first primer (SEQ ID NO: 3)
1.0 μM second primer (SEQ ID NO: 4)
12.8U AMV reverse transcriptase 166U T7 RNA polymerase 0.33 mM irradiated or unirradiated dATP or NB-dATP (prepared with (1))
(3) After adding the initiator in the same manner as in Example 1 (3), the measurement was carried out in the same manner as in Example 1 (4) to determine the detection time.

Table 3 shows the results when the dATP solution or NB-dATP solution was not irradiated with ultraviolet light or was irradiated for 1 minute to 40 minutes. The NB-dATP solution was not irradiated with ultraviolet light or irradiated for 1 minute to 10 minutes. Table 4 shows the results of each of these cases. When dATP was added to the reaction solution, there was no effect on the TRC reaction depending on the presence or absence of ultraviolet light irradiation (Table 3). On the other hand, when NB-dATP was added to the reaction solution, the effect on the TRC reaction changed depending on the ultraviolet light irradiation time. When not irradiated with ultraviolet light and the irradiation time is 0.017 minutes or less, the unmodified dATP is TRC.
The TRC reaction did not proceed because the amount required for the reaction was not contained in the reaction solution (Tables 3 and 4). When ultraviolet light was irradiated for 0.5 to 5 minutes, the TRC reaction proceeded due to the unmodified dATP generated by the irradiation, and the detection time was equivalent to that when the unmodified dATP was added (Table 3). And Table 4). When irradiated with ultraviolet light for 10 minutes or more, the TRC reaction is inhibited by the photodegradable protecting group (2-nitrobenzyl group) generated by the irradiation, and the detection time is slower than when unmodified dATP is added. (Tables 3 and 4).

Example 4 Optical control of TRC reaction using NB-dATP A TRC reaction solution to which NB-dATP was added instead of dATP was used, and the control effect of the TRC reaction by light irradiation was investigated.

(1) After dispensing 12 μL of the reaction solution having the following composition into a 0.5 mL volume PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), 2 μL of the RNA sample prepared in Example 1 (1) was added. ..

Composition of reaction solution: The concentration is the final concentration after addition of the initiator described below (in 20 μL) 66 mM Tris-HCl buffer (pH 8.36).
0.33 mM dCTP, dGTP, dTTP, respectively
2.0 mM ATP, CTP, GTP, UTP respectively
3.3 mM ITP
150 mM trehalose 50 nM molecular beacon probe (SEQ ID NO: 2)
1.0 μM first primer (SEQ ID NO: 3)
1.0 μM second primer (SEQ ID NO: 4)
12.8U AMV reverse transcriptase 166U T7 RNA polymerase 0.33 mM dATP or NB-dATP
(2) The above reaction solution was kept warm at 46 ° C. for 3 minutes, and then 6 μL of the initiator having the composition described in Example 1 (3) was added. When keeping warm, start keeping warm by shifting the irradiation time of ultraviolet light (wavelength 365 nm, intensity 700 μW / cm ² ) to a warmer at 46 ° C, so that the irradiation of ultraviolet light to the PCR tube under consideration ends at the same time. bottom.

(3) After the completion of the ultraviolet light irradiation, the measurement was carried out in the same manner as in Example 1 (4), and the detection time was determined. The detection time of 0 minutes is the time when the thermometer is moved to the fluorescence spectrophotometer.

The results are shown in Table 5. When not irradiated with ultraviolet light, the TRC reaction did not proceed because there was no unmodified dATP in the reaction solution. When the reaction solution was irradiated with ultraviolet light for 1 minute, the reaction time was as slow as about 25 minutes due to the presence of unmodified dATP in the reaction solution due to the small amount of the unmodified dATP. When the ultraviolet light was irradiated for 1.5 to 4 minutes, the TRC reaction proceeded due to the unmodified dATP generated by the irradiation, and the UV light irradiation time could be detected in about 11 minutes. When ultraviolet light was irradiated for 5 minutes or more, the TRC reaction was inhibited by the photodegradable protecting group (2-nitrobenzyl group) generated by the irradiation, and the TRC reaction was not detected. From this result, the TRC reaction can be controlled by light by modifying at least one of the deoxynucleoside triphosphates contained in the reaction solution (deoxyadenosine triphosphate in this example) with a photodegradable protecting group. I understand.

Example 5 Effect of photodegradable protecting group on TRC reaction (Part 2)
In Examples 2 to 4, 2-nitrobenzyl group was used as a photodegradable protecting group, but in this experiment, the effect of 3- (diethylamino) benzyl group, which can also be used as a photodegradable protecting group, on the TRC reaction was observed. Examined.

(1) According to Synthesis Examples 2 to 3, the 3'-terminal hydroxy group of adenosine was modified with a 3- (diethylamino) benzyl group as a photodegradable protecting group, and 3'-O- [3- (diethylamino) benzyl]- 2'-deoxyadenosine was synthesized.

(2) The solution containing 3'-O- [3- (diethylamino) benzyl] -2'-deoxyadenosine synthesized in (1) was dissolved in DMSO to a constant concentration.

(3) Prepared in Example 1 (1) after dispensing 8 μL of the reaction solution having the composition described in Example 2 (3) into a 0.5 mL volume PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI). 2 μL of the RNA sample was added.

(4) The above reaction solution was kept warm at 46 ° C. for 3 minutes, and then 10 μL of an initiator having the following composition was added.

Initiator composition: The concentration is the final concentration after addition of the initiator (in 20 μL) 18.4 mM magnesium chloride 90.0 mM potassium chloride 0.1% (w / v) Tween 20 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
2.5% (w / v) Glycerol 10% (v / v) DMSO (containing 0 to 200 μM as a photodegradable protecting group, prepared in (2))
(5) The detection time was determined by measurement in the same manner as in Example 1 (4).

The results are shown in Table 6. It can be seen that when a diethylaminobenzyl group is used as the photodegradable protecting group, there is no effect on the TRC reaction as long as the concentration is at least 200 μM.

Example 6 Photocontrol of TRC reaction using photodegradable protecting group-modified primer (1) Using the solid-phase-supported thymidine derivative obtained in Synthesis Example 11, 3'-terminal (49th) thymidine 3 A first primer consisting of the nucleotide sequence shown in SEQ ID NO: 7 in which the hydroxy group at the'position'was modified with a photodegradable protecting group (hereinafter, also simply referred to as "photodegradable protecting group-modifying primer") was synthesized (hereinafter, also referred to as "photoresolvable protecting group modifying primer"). Outsourced to Gene Design). Of the first primer consisting of the sequence shown in SEQ ID NO: 7, 28 bases on the 5'end side are the T7 promoter sequence (SEQ ID NO: 6). The synthetic product was purified by HPLC, and after confirming the molecular weight with an electrospray ionization mass spectrometer (ESI-MS), it was diluted to 100 μM with TE buffer and stored at −20 ° C.

(2) 8 μL of the reaction solution having the following composition was dispensed into a 0.5 mL volume PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI).

Composition of reaction solution: The concentration is the final concentration of 0.1% (w / v) after the addition of the initiator described later (in 20 μL) Tween 20 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
66 mM Tris-HCl buffer (pH 8.36)
2.5% (w / v) glycerol 0.33 mM dATP, dCTP, dGTP, dTTP, respectively
2.0 mM ATP, CTP, GTP, UTP respectively
3.3 mM ITP
94.5 mM trehalose 50 nM molecular beacon probe (SEQ ID NO: 2)
1.0 μM second primer (SEQ ID NO: 4)
45.4U AMV Reverse Transcriptase 100UT7 RNA polymerase (3) Photodegradable protecting group-modified primer solution synthesized in (1) prepared to 10 μM using water for injection, or (without modification by photodegradable protecting group) ) 2 μL of the first primer solution consisting of the nucleotide sequence shown in SEQ ID NO: 7 was added to the above reaction solution without irradiation with ultraviolet light (ultraviolet light irradiation time: 0 minutes) (after the addition of the initiator described later). 1 μM as the final concentration).

(4) The 10 μM photodegradable protecting group-modified primer solution prepared in (3) was subjected to ultraviolet light (wavelength 365 nm, intensity 1 mW) using an ultraviolet light irradiation device (Omnicure LX405S UV LED spot curing device, manufactured by Sanei Tech Co., Ltd.). / Cm ² ) was irradiated for 1 minute or 10 minutes. 2 μL of the irradiated primer solution was added to the above reaction solution.

(5) Standard RNA (SEQ ID NO: 1) TE buffer (0.01% (w / v) cholate added) after diluted to ¹⁰⁵ copies / 2 [mu] L using the above (3) and (4) 2 μL was added to the reaction solution to which the primer solution was added, which was prepared in 1.

(6) After the addition of the standard RNA, the reaction solution was kept warm at 46 ° C. for 3 minutes, and 8 μL of the initiator having the composition described in Example 1 (3), which was also kept warm at 46 ° C. for 3 minutes, was added.

(7) The detection time was determined by measurement in the same manner as in Example 1 (4).

The results are shown in Table 7. From the result of the ultraviolet irradiation time of 0 minutes, when the photodegradable protecting group-modified primer synthesized in (1) was used as the first primer, it was compared with the case where the first primer not modified by the photodegradable protecting group was used. However, it can be seen that the detection time is delayed by about 1 minute. From this, it can be seen that the TRC reaction can be inhibited by modifying the deoxyribonucleotide at the 3'end of the primer with a photodegradable protecting group. On the other hand, even if the photodegradable protecting group-modified primer synthesized in (1) is used as the first primer, when the protective group is decomposed by irradiating with ultraviolet light for 1 minute or 10 minutes, the unmodified first primer is used. The detection time was equivalent to that when one primer was used. From this, it can be seen that the TRC reaction can be controlled by light by modifying the deoxyribonucleotide at the 3'end of the primer with a photodegradable protecting group.

Example 7 Effect of TRC reaction on TRC reactivity due to light control In Example 6, only the first primer was irradiated with ultraviolet light, but in this experiment, the reaction solution containing the first primer was exposed to ultraviolet light. Was irradiated, and the effect of the irradiation on the TRC reaction was investigated.

(1) 8 μL of the reaction solution having the composition described in Example 6 (2) was dispensed into a 0.5 mL volume PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI).

(2) A 10 μM photodegradable protecting group-modified primer (SEQ ID NO: 7) solution or a first primer (SEQ ID NO: 7) solution (without modification by a photodegradable protecting group) prepared in Example 6 (3). , 2 μL each was added to the above reaction solution (1 μM as the final concentration after the addition of the initiator described later).

(3) after diluted to ¹⁰⁵ copies / 2 [mu] L using standard RNA (SEQ ID NO: 1) TE buffer (0.01% (w / v) cholate added) were prepared in the above (2), 2 μL was added to the reaction solution to which the primer solution was added.

(4) After adding the standard RNA, the reaction solution was kept warm at 46 ° C. for 3 minutes, and 8 μL of the initiator having the composition according to Example 1 (3) was similarly kept warm at 46 ° C. for 3 minutes, and then ultraviolet light (4). The wavelength was 365 nm and the intensity ^{was 1 mw / cm 2} ) for 1 minute.

(5) The detection time was determined by measurement in the same manner as in Example 1 (4).

The results are shown in Table 8. Even if the entire reaction solution was irradiated with ultraviolet light, the detection time was the same as in Example 6. From this, it can be seen that even if the entire reaction solution is irradiated with ultraviolet light, there is no effect on the TRC reaction.

Claims (8)

An enzyme having RNA-dependent DNA polymerase activity, an enzyme having DNA-dependent DNA polymerase activity, an enzyme having ribonuclease H (RNase H) activity, and D
An enzyme having NA-dependent RNA polymerase activity, a first primer having a sequence complementary to a part of the target nucleic acid, and a second primer having a sequence homologous to a part of the target nucleic acid.
An amplification reagent for the target nucleic acid, comprising ribonucleoside triphosphate and deoxynucleoside triphosphate (provided that either the first primer or the second primer has the 5'terminal side thereof. The primer sequence of the enzyme having RNA polymerase activity is added).
Photocontrolled amplification reagent of the target nucleic acid satisfying at least one of the following <1> and <2>:
<1> The primer represented by the following general formula (0), wherein the first and / or the second primer is a primer in which the deoxyribonucleotide at the 3'end of the primer is modified with a photodegradable protecting group.

In the general formula (0), Z represents an oligodeoxyribonucleotide constituting the first or second primer, and X represents a nucleobase according to any of the following formulas (2) to (5).

And Y is an o-nitrobenzyl group represented by the following general formula (7).

(In the general formula (7), X ¹ , X ² , X ³ and X ⁴ independently represent a hydrogen atom and an alkyl group or a hydroxymethyl group having 1 to 6 carbon atoms, respectively.)
Alternatively, it represents a 3- (dialkylamino) benzyl group represented by the following general formula (6).

(In general formula (6), R ¹ and R ² each independently represent an alkyl group having 1 to 8 carbon atoms, or R ¹ and R ² are integrated with the nitrogen atom to which they are bonded to 5 to 7. A heterocyclic ring of members may be formed. R ³ and R ⁴ independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a phenyl group.)
<2> At least one of deoxyadenosine triphosphate, deoxythymidine triphosphate, deoxyguanosine triphosphate and deoxycytidine triphosphate constituting the deoxynucleoside triphosphate is a photodegradable protective group. A modified photoresponsive deoxynucleoside triphosphate represented by the following general formula (1).

In the general formula (1), X represents any of the nucleobases of the following formulas (2) to (5).

And Y is an o-nitrobenzyl group represented by the following general formula (7).

(In the general formula (7), X ¹ , X ² , X ³ and X ⁴ independently represent a hydrogen atom and an alkyl group or a hydroxymethyl group having 1 to 6 carbon atoms, respectively.)
Alternatively, it represents a 3- (dialkylamino) benzyl group represented by the following general formula (6).

(In general formula (6), R ¹ and R ² each independently represent an alkyl group having 1 to 8 carbon atoms, or R ¹ and R ² are integrated with the nitrogen atom to which they are bonded to 5 to 7. A heterocyclic ring of members may be formed. R ³ and R ⁴ independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a phenyl group.) The photocontrolled amplification reagent for a target nucleic acid according to claim 1, wherein the target nucleic acid is DNA and / or RNA. A method for amplifying a target nucleic acid using the photo-controlled amplification reagent for the target nucleic acid according to claim 1 or 2, wherein the amplification reaction for the target nucleic acid is started by irradiation with light. The method for amplifying a target nucleic acid according to claim 3, wherein the target nucleic acid is RNA. The method for amplifying RNA according to claim 4, wherein the method for amplifying RNA is any of the NASBA method, the TMA method, and the TRC method. The method for amplifying RNA according to claim 5, wherein the method for amplifying RNA is the TRC method. The target nucleic acid comprising the photocontrolled amplification reagent according to claim 1 or 2 and an oligonucleotide probe whose fluorescence characteristics change when a complementary double strand is formed with a part of the amplified target nucleic acid as compared with that before the formation. Detection reagent. A method for amplifying a target nucleic acid using the target nucleic acid detection reagent according to claim 7, wherein the amplification reaction of the target nucleic acid is started by light irradiation to detect the amplified target nucleic acid. Detection method.